Application of functional genomics in allergy and clinical immunology.
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Computational Biology↗
Biomedical subjects
J H Shelhamer
Publications and source records attributed to J H Shelhamer.
p11 expression in human bronchial epithelial cells is increased by nitric oxide in a cGMP-dependent pathway involving protein kinase G activation.
The effect of nitric oxide on p11 expression was studied in an immortalized human bronchial epithelial cell line (BEAS-2B cells). Three nitric oxide donors were used: spermine NONOate (SP), (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), and S-nitrosoglutathione (SNOG). All three nitric oxide donors had similar effects resulting in dose-dependent and time-dependent accumulation of p11 protein and an increase of steady-state p11 mRNA. Studies using a reporter gene containing the region from -1499 to +89 of the p11 promoter demonstrated an increase in transcriptional activity after stimulation with NO donors for 4 h. These effects were abolished at the promoter and protein level using protein kinase G inhibitors (KT5823 and R(p)-8-pCPT-cGMPS). Incubation of transfected cells with a cell permeable cGMP analogue (8-Br-cGMP) resulted in a dose-related increase of promoter activity. An electrophoretic mobility shift assay of nuclear proteins extracted from BEAS-2B cells identified an AP-1 site located at -82 to -77 of the p11 promoter region as an NO- and cGMP- dependent response element. These data were confirmed using a c-jun dominant negative mutant vector and a c-jun expression plasmid. Therefore, we conclude that nitric oxide-induced p11 expression in human bronchial epithelial cells is mediated at least in part through increased binding of activator protein one to the p11 promoter.
Alkaloids↗
Nitric oxide transport on sickle cell hemoglobin: where does it bind?
We have recently reported that nitric oxide inhalation in individuals with sickle cell anemia increases the level of NO bound to hemoglobin, with the development of an arterial-venous gradient, suggesting delivery to the tissues. A recent model suggests that nitric oxide, in addition to its well-known reaction with heme groups, reacts with the beta-globin chain cysteine 93 to form S-nitrosohemoglobin (SNO-Hb) and that SNO-Hb would preferentially release nitric oxide in the tissues and thus modulate blood flow. However, we have also recently determined that the primary NO hemoglobin adduct formed during NO breathing in normal (hemoglobin A) individuals is nitrosyl (heme)hemoglobin (HbFeIINO), with only a small amount of SNO-Hb formation. To determine whether the NO is transported as HbFeIINO or SNO-Hb in sickle cell individuals, which would have very different effects on sickle hemoglobin polymerization, we measured these two hemoglobin species in three sickle cell volunteers before and during a dose escalation of inhaled NO (40, 60, and 80 ppm). Similar to our previous observations in normal individuals, the predominant species formed was HbFeIINO, with a significant arterial-venous gradient. Minimal SNO-Hb was formed during NO breathing, a finding inconsistent with significant transport of NO using this pathway, but suggesting that this pathway exists. These results suggest that NO binding to heme groups is physiologically a rapidly reversible process, supporting a revised model of hemoglobin delivery of NO in the peripheral circulation and consistent with the possibility that NO delivery by hemoglobin may be therapeutically useful in sickle cell disease.
Anemia, Sickle Cell↗
Disorders of ciliary motility.
Clearance of mucus and other debris from the airways is achieved by 3 main mechanisms: mucociliary activity, coughing, and alveolar clearance. Disorders of ciliary structure or function results in impaired clearance, and result in chronic sinopulmonary disease manifested as chronic sinusitis, otitis media, nasal polyposis, and ultimately bronchiectasis. In addition, situs inversus, dextrocardia, and infertility can be associated with dysfunctional ciliary activity. The term primary ciliary dyskinesia has been proposed for the spectrum of these diseases. The term Kartagener syndrome applies to this syndrome when accompanied by infertility and dextrocardia or situs inversus. The more common types of ciliary dysmotility syndromes are characterized by missing dynein arms, central microtubule pairs, inner sheath, radial spokes, or nexin links. In addition to structural defects within the cilia, disordered ciliary beating and disordered ciliary arrays on epithelial cell surfaces have been described in this syndrome. Treatment includes rigorous lung physiotherapy, prophylactic and organism-specific antibiotics, and immunization against common pulmonary pathogens. Late stages of the disease may require surgical intervention for bronchiectasis or lung transplant for end-stage lung disease.
Adult↗
Prostaglandin E(2)-induced interleukin-6 release by a human airway epithelial cell line.
Human airway epithelial cell release of interleukin (IL)-6 in response to lipid mediators was studied in an airway cell line (BEAS-2B). Prostaglandin (PG) E(2) (10(-7) M) treatment caused an increase in IL-6 release at 2, 4, 8, and 24 h. IL-6 release into the culture medium at 24 h was 3,396 +/- 306 vs. 1,051 +/- 154 pg/ml (PGE(2)-treated cells vs. control cells). PGE(2) (10(-7) to 10(-10) M) induced a dose-related increase in IL-6 release at 24 h. PGF(2 alpha) (10(-6) M) treatment caused a similar effect to that of PGE(2) (10(-7) M). PGE(2) analogs with relative selectivity for PGE(2) receptor subtypes were studied. Sulprostone, a selective agonist for the EP-3 receptor subtype had no effect on IL-6 release. 11-Deoxy-16,16-dimethyl-PGE(2), an EP-2/4 agonist, and 17-phenyl trinor PGE(2), an agonist selective for the EP-1 > EP-3 receptor subtype (10(-6) to 10(-8) M), caused dose-dependent increases in IL-6 release. 8-Bromo-cAMP treatment resulted in dose-related increases in IL-6 release. RT-PCR of BEAS-2B cell mRNA demonstrated mRNA for EP-1, EP-2, and EP-4 receptors. After PGE(2) treatment, increases in IL-6 mRNA were noted at 4 and 18 h. Therefore, PGE(2) increases airway epithelial cell IL-6 production and release.
8-Bromo Cyclic Adenosine Monophosphate↗
Effects of inhaled nitric oxide on regional blood flow are consistent with intravascular nitric oxide delivery.
Nitric oxide (NO) may be stabilized by binding to hemoglobin, by nitrosating thiol-containing plasma molecules, or by conversion to nitrite, all reactions potentially preserving its bioactivity in blood. Here we examined the contribution of blood-transported NO to regional vascular tone in humans before and during NO inhalation. While breathing room air and then room air with NO at 80 parts per million, forearm blood flow was measured in 16 subjects at rest and after blockade of forearm NO synthesis with N(G)-monomethyl-L-arginine (L-NMMA) followed by forearm exercise stress. L-NMMA reduced blood flow by 25% and increased resistance by 50%, an effect that was blocked by NO inhalation. With NO inhalation, resistance was significantly lower during L-NMMA infusion, both at rest and during repetitive hand-grip exercise. S-nitrosohemoglobin and plasma S-nitrosothiols did not change with NO inhalation. Arterial nitrite levels increased by 11% and arterial nitrosyl(heme)hemoglobin levels increased tenfold to the micromolar range, and both measures were consistently higher in the arterial than in venous blood. S-nitrosohemoglobin levels were in the nanomolar range, with no significant artery-to-vein gradients. These results indicate that inhaled NO during blockade of regional NO synthesis can supply intravascular NO to maintain normal vascular function. This effect may have application for the treatment of diseases characterized by endothelial dysfunction.
Administration, Inhalation↗
Role of circulating nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans.
To determine the relative contributions of endothelial-derived nitric oxide (NO) vs. intravascular nitrogen oxide species in the regulation of human blood flow, we simultaneously measured forearm blood flow and arterial and venous levels of plasma nitrite, LMW-SNOs and HMW-SNOs, and red cell S-nitrosohemoglobin (SNO-Hb). Measurements were made at rest and during regional inhibition of NO synthesis, followed by forearm exercise. Surprisingly, we found significant circulating arterial-venous plasma nitrite gradients, providing a novel delivery source for intravascular NO. Further supporting the notion that circulating nitrite is bioactive, the consumption of nitrite increased significantly with exercise during the inhibition of regional endothelial synthesis of NO. The role of circulating S-nitrosothiols and SNO-Hb in the regulation of basal vascular tone is less certain. We found that low-molecular-weight S-nitrosothiols were undetectable and S-nitroso-albumin levels were two logs lower than previously reported. In fact, S-nitroso-albumin primarily formed in the venous circulation, even during NO synthase inhibition. Whereas SNO-Hb was measurable in the human circulation (brachial artery levels of 170 nM in whole blood), arterial-venous gradients were not significant, and delivery of NO from SNO-Hb was minimal. In conclusion, we present data that suggest (i) circulating nitrite is bioactive and provides a delivery gradient of intravascular NO, (ii) S-nitroso-albumin does not deliver NO from the lungs to the tissue but forms in the peripheral circulation, and (iii) SNO-Hb and S-nitrosothiols play a minimal role in the regulation of basal vascular tone, even during exercise stress.
Adult↗
Relative role of heme nitrosylation and beta-cysteine 93 nitrosation in the transport and metabolism of nitric oxide by hemoglobin in the human circulation.
To quantify the reactions of nitric oxide (NO) with hemoglobin under physiological conditions and to test models of NO transport on hemoglobin, we have developed an assay to measure NO-hemoglobin reaction products in normal volunteers, under basal conditions and during NO inhalation. NO inhalation markedly raised total nitrosylated hemoglobin levels, with a significant arterial-venous gradient, supporting a role for hemoglobin in the transport and delivery of NO. The predominant species accounting for this arterial-venous gradient is nitrosyl(heme)hemoglobin. NO breathing increases S-nitrosation of hemoglobin beta-chain cysteine 93, however only to a fraction of the level of nitrosyl(heme)hemoglobin and without a detectable arterial-venous gradient. A strong correlation between methemoglobin and plasma nitrate formation was observed, suggesting that NO metabolism is a primary physiological cause of hemoglobin oxidation. Our results demonstrate that NO-heme reaction pathways predominate in vivo, NO binding to heme groups is a rapidly reversible process, and S-nitrosohemoglobin formation is probably not a primary transport mechanism for NO but may facilitate NO release from heme.
Administration, Inhalation↗
Tumor necrosis factor alpha stimulation of human Clara cell secretory protein production by human airway epithelial cells.
Clara cell secretory protein (CCSP) or uteroglobin/CC10 is a product of epithelial cells in a variety of organs including the lung. CCSP has anti-inflammatory properties and may act as an inhibitor of secretory phospholipase A2's. Tumor necrosis factor alpha (TNF-alpha) is capable of inducing the expression of gene products including a variety of cytokines and chemokines in the airway epithelium that may upregulate the airway inflammatory response. Therefore, it was of interest to determine whether this proinflammatory cytokine might also induce the production of a counterregulatory protein such as CCSP, which might modulate the inflammatory response in the airway. Normal human tracheobronchial epithelial cells in primary culture and a human bronchial epithelial cell line (BEAS-2B) were studied. CCSP mRNA levels in BEAS-2B cells were detected by ribonuclease protection assay. CCSP mRNA levels increased in response to TNF-alpha (20 ng/mL) stimulation after 8-36 h, with the peak increase at 18 h. Immunoblotting of CCSP released from BEAS-2B cells into the culture media demonstrated that TNF-alpha induced the synthesis and secretion of CCSP over 8 to 18 h. Similarly, TNF stimulated the release of CCSP from human tracheobronchial epithelial cells in primary culture at 8 and 18 h. The CCSP reporter gene including 801 bases 5' of the transcription start site did not increase transcriptional activity in response to TNF-alpha stimulation. A CCSP mRNA half-life assay indicated that TNF-alpha induced increases in CCSP mRNA at least in part at a posttranscriptional level. Therefore, TNF-alpha induces airway epithelial cell expression of human CCSP and may modulate airway inflammatory responses in this manner.
Animals↗
Retinoic acid reduces p11 protein levels in bronchial epithelial cells by a posttranslational mechanism.
p11 is a member of the S100 family of proteins, is the cellular ligand of annexin II, and interacts with the carboxyl region of 85-kDa cytosolic phospholipase A(2) (cPLA(2)), inhibiting cPLA(2) activity and arachidonic acid (AA) release. We studied the effect of retinoic acid (RA) on PLA(2) activity in human bronchial epithelial cells and whether p11 contributes to these effects. The addition of 10(-6) M RA resulted in reduced p11 protein levels at 4 days, with the greatest effect observed on days 6 and 7. This effect was dose related (10(-6) to 10(-9) M). RA treatment (10(-6) M) had no effect on cPLA(2) protein levels. p11 mRNA levels were unchanged at 6 and 8 days of treatment (correlating with maximum p11 protein reduction). Treatment with RA reduced p11 levels in control cells and in cells transfected with a p11 expression vector, suggesting a posttranslational mechanism. Lactacystin (10(-6) M), an inhibitor of the human 26S proteasome, blocked the decrease in p11 observed with RA treatment. Compared with control cells (n = 3), RA-treated cells (n = 3) had significantly increased AA release after treatment with the calcium ionophore A-23187 (P = 0.006). Therefore, RA reduces p11 protein expression and increases PLA(2) activity and AA release.
Acetylcysteine↗
Pneumocystis pneumonia: miles to go.
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AIDS-Related Opportunistic Infections↗
Dexamethasone alters arachidonate release from human epithelial cells by induction of p11 protein synthesis and inhibition of phospholipase A2 activity.
The effect of the glucocorticosteroid, dexamethasone, on arachidonic acid (AA) release and on protein levels of p11 and cytosolic phospholipase A2 (cPLA2) was studied in two epithelial cell lines, HeLa cells and BEAS-2B cells. Dexamethasone treatment of HeLa cells and BEAS-2B cells increased cellular p11 protein and mRNA levels in a time- and dose-dependent manner. It had little effect on levels of cPLA2 protein. In order to determine if increased p11 protein expression resulted in increased interaction between p11 and cPLA2, anti-cPLA2 antibodies were used to immunoprecipitate p11.cPLA2 complexes and Western blots of the immunoprecipitate were used to detect p11. In cells treated with dexamethasone, more p11 was detected in the anti-cPLA2 immunoprecipitate compared with control cells. Dexamethasone treatment of HeLa cells prelabeled with [3H]AA decreased the release of [3H]AA under basal conditions and after stimulation with the calcium ionophore A23187 (10(-6) M). In order to determine if altering the p11 protein levels in HeLa cells independent of glucocorticosteroid treatment could also produce an effect on [3H]AA release, cells were stably transfected with plasmids expressing either p11 antisense mRNA or p11 mRNA. Cloned HeLa cells expressing p11 antisense mRNA exhibited less cellular p11 protein compared with control cells and greater [3H]AA release compared with cells transfected with a control vector. Cloned HeLa cells stably transfected with a p11 expression vector exhibited increased p11 cellular protein and diminished [3H]AA release under basal conditions and in response to A23187. Therefore, dexamethasone alteration of epithelial cell AA release may be due in part to induction of p11 protein expression.
Annexin A2↗
Pneumocystis carinii major surface glycoprotein induces interleukin-8 and monocyte chemoattractant protein-1 release from a human alveolar epithelial cell line.
BACKGROUND: The major surface glycoprotein (MSG) is an abundant, immunogenic glycoprotein located on the surface of Pneumocystis carinii. Little is known about the proinflammatory effects of MSG. DESIGN: We have investigated the effect of human MSG on the secretion of the chemokines interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) from an alveolar epithelial cell line (A549). RESULTS: Incubation of A549 cells with MSG in concentrations from 0.4 to 10 microg mL-1 for 24 h caused dose-dependent increases in IL-8 release (3.4-fold above control, P < 0.01). Time course experiments showed increases in IL-8 release at 4 h, 8 h and 24 h compared with control cultures (all P < 0.01). There was a minor (13%) dose- and time-related increase in MCP-1 release at 24 h (P = 0.02). Co-incubation of MSG with mannan or beta-glucan decreased IL-8 release by 48% and 42% respectively, suggesting that MSG stimulates A549 cells in part through carbohydrate moieties. Dexamethasone significantly inhibited MSG-induced IL-8 release in concentrations of 10-6-10-8 mol L-1 compared with control experiments (P < 0.01). Ribonuclease protection assays for steady-state IL-8 mRNA showed that increases in response to MSG stimulation occurred by 4 h and persisted throughout 8 h of stimulation. CONCLUSION: These findings suggest that MSG can alter alveolar epithelial cytokine release and may be capable of modulating the local inflammatory response in this manner.
Cell Line↗
The acute chest syndrome in sickle cell disease. Possible role of nitric oxide in its pathophysiology and treatment.
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Acute Disease↗
Inhaled nitric oxide augments nitric oxide transport on sickle cell hemoglobin without affecting oxygen affinity.
Nitric oxide (NO) inhalation has been reported to increase the oxygen affinity of sickle cell erythrocytes. Also, proposed allosteric mechanisms for hemoglobin, based on S-nitrosation of beta-chain cysteine 93, raise the possibility of altering the pathophysiology of sickle cell disease by inhibiting polymerization or by increasing NO delivery to the tissue. We studied the effects of a 2-hour treatment, using varying concentrations of inhaled NO. Oxygen affinity, as measured by P(50), did not respond to inhaled NO, either in controls or in individuals with sickle cell disease. At baseline, the arterial and venous levels of nitrosylated hemoglobin were not significantly different, but NO inhalation led to a dose-dependent increase in mean nitrosylated hemoglobin, and at the highest dosage, a significant arterial-venous difference emerged. The levels of nitrosylated hemoglobin are too low to affect overall hemoglobin oxygen affinity, but augmented NO transport to the microvasculature seems a promising strategy for improving microvascular perfusion.
Administration, Inhalation↗
Polypoid lesions of airways: early experience with computer-assisted detection by using virtual bronchoscopy and surface curvature.
PURPOSE: To test the application of a technique developed by the authors for the computer-assisted diagnosis of polypoid airway lesions from surface rendered virtual bronchoscopic reconstructions. MATERIALS AND METHODS: A computer algorithm was developed to detect polypoid airway lesions by means of segmentation of the bronchial surface with curvature classification. This method was tested with a bronchial phantom, five cadaveric lung specimens, and virtual bronchoscopic studies in 16 patients. RESULTS: For the patient studies, the sensitivity and specificity of the method were 47%-88% and 58%-89%, respectively, depending on the value of an adjustable parameter (the mean curvature threshold). The sensitivity increased (by 20% to 34%) when only lesions larger than 5 mm in diameter were considered. CONCLUSION: With this method, polypoid airway lesions can be detected automatically, although false-positive diagnoses present an important limitation.
Adult↗
Interferon-gamma stimulates human Clara cell secretory protein production by human airway epithelial cells.
Clara cell secretory protein (CCSP) is an inhibitor of secretory phospholipase A2. It is produced by airway epithelial cells and is present in airway secretions. Because interferon (IFN)-gamma can induce gene expression in airway epithelial cells and may modulate the inflammatory response in the airway, it was of interest to study the effect of this cytokine on epithelial cell CCSP mRNA expression and CCSP protein synthesis. A human bronchial epithelial cell line (BEAS-2B) was used for this study. CCSP mRNA was detected by ribonuclease protection assay. IFN-gamma was found to increase CCSP mRNA expression in a time- and dose-dependent manner. The CCSP mRNA level increased after IFN-gamma (300 U/ml) treatment for 8-36 h, with the peak increase at 18 h. Immunobloting of CCSP protein also demonstrated that IFN-gamma induced the synthesis and secretion of CCSP protein in a time-dependent manner. Nuclear run-on, CCSP reporter gene activity assay, and CCSP mRNA half-life assay demonstrated that IFN-gamma-induced increases in CCSP gene expression were mediated, at least in part, at the posttranscriptional level. The present study demonstrates that IFN-gamma can induce increases in steady-state mRNA levels and protein synthesis of human CCSP protein in airway epithelial cells and may modulate airway inflammatory responses in this manner.
Bronchi↗
Tumor necrosis factor-alpha stimulates human Clara cell secretory protein production by human airway epithelial cells.
Clara cell secretory protein (CCSP), or CC10, is an inhibitor of secretory phospholipase A2 which may be produced by phagocytic cells and by a variety of other cells in the airway. Tumor necrosis factor-alpha (TNF-alpha) is capable of activating phospholipases and inducing the expression of a variety of genes in the airway epithelium which may modulate the airway inflammatory response. Therefore, it was of interest to determine whether this proinflammatory cytokine could induce the production of a counterregulatory protein such as CCSP which might modulate the production of eicosanoid mediators in the airway. Using a human bronchial epithelial cell line (BEAS-2B), CCSP messenger RNA (mRNA) levels were detected by ribonuclease protection assay. TNF treatment of these cells increased CCSP mRNA levels in a time- and dose-dependent manner. The CCSP mRNA level increased in response to TNF-alpha (20 ng/ml) stimulation after 8 to 36 h with the peak increase at 18 h. Immunoblotting of CCSP protein released into the culture media demonstrated that TNF-alpha induced the synthesis and secretion of CCSP protein in a time-dependent manner over 8 to 18 h. The results of a CCSP reporter gene activity assay, nuclear run-on assay, and CCSP mRNA half-life assay indicated that the TNF-alpha-induced increases in CCSP gene expression are regulated at the post-transcriptional level. We conclude that TNF-alpha induces airway epithelial cell expression of human CCSP protein and may modulate airway inflammatory responses in this manner.
Bronchi↗